One major goal in transplantation immunology is to develop new immunosuppressive agents with the potential to induce donor-specific tolerance and thus avoid increased risk of infection and cancer in transplant recipients. Considerable effort has been spent on the study of costimulation-targeting therapies, as these regimens successfully induce permanent acceptance of tissues such as heart grafts and pancreatic islets in rodents. However, we and others have recently shown that infections at the time of transplantation potently antagonize the ability of costimulation-blockade therapies to achieve donor-specific tolerance. Microorganisms contain molecular patterns that are recognized by Toll-like receptors (TLRs) expressed on host and donor cells. Our results indicate that engagement of a single TLR at the time of cardiac transplantation is sufficient to prevent graft acceptance induced by anti-CD154 donor-specific transfusion (DST) or CTLA-4-Ig in mice. Understanding the mechanisms by which TLR signaling opposes pathways of transplantation tolerance is essential to develop novel therapies likely to be successful in the clinic, in patients constantly exposed to both commensal and pathogenic microorganisms. Induction and maintenance of cardiac transplantation tolerance by anti-CD154/DST is known to depend on regulatory T cells (Tregs). We have shown that anti-CD154 promotes intragraft accumulation of Tregs and development of dominant regulation in the form of linked-suppression. These phenomena are both antagonized by the TLR9 agonist CpG-B. Our preliminary results have shown that CpG prevents Treg recruitment to cardiac allografts and inhibits the ability of anti-CD154 to achieve a high Treg to effector T cell (Teff) ratio in the graft. Treg:Teff ratio in target tissues is emerging as a critical parameter controlling the outcome of immune responses in vivo, but signals determining this ratio are not well understood. Our results support the central hypothesis that TLR signaling controls the Treg/Teff ratio in the allograft and the consequent fate of transplanted organs. Our preliminary results suggest there may be effects of TLR signaling on both Tregs and Teffs. In this application, we will study the mechanisms by which TLR signaling opposes transplantation tolerance. This will be performed in the context of the following specific aims. Specific Aim 1. To investigate the mechanisms by which CpG limits intragraft Treg accumulation during prevention of transplantation tolerance. Specific Aim 2. To delineate nTreg-independent effects of TLR signaling on graft-reactive Teffs and APCs during the prevention of transplantation tolerance. Completion of these aims should reveal new consequences of TLR signaling in vivo and should yield a deeper understanding of different facets of the interplay between innate and adaptive immune responses that may negatively impact establishment of transplantation tolerance. Project Narrative: The interplay between innate and adaptive immune system determines the fate of an immune response. We have recently shown that microbial products that signal via toll-like receptors (TLRs) can prevent the induction of transplantation tolerance. In this application, we propose to investigate the mechanisms by which TLR signaling antagonizes tolerogenic signals.